title: Quantitative study of protein-protein interactions in live cell by dual-color fluorescence correlation spectroscopy.
abstract: Dual-color FCS is a powerful method to monitor protein-protein interactions in living cells. The main idea is based on the cross-correlation analysis of temporal fluorescence intensity fluctuations of two fluorescent proteins to obtain their co-diffusion and relative concentration. But, when performing these experiments, the spectral overlap in the emission of the two colors produces an artifact that corrupts the cross-correlation data: spectral bleed-through. We have shown that problems with cross talk are overcome with Fluorescence Lifetime Correlation Spectroscopy (FLCS). FLCS applied to dual-color cross-correlation, utilizing for example eGFP and mCherry fluorescent proteins, allows the determination of protein-protein interactions in living cells without the need of spectral bleed-through calibration. Here, we present in detail how this methodology can be implemented using a commercial setup (Microtime from PicoQuant, SP8 SMD from Leica or any conventional confocal with PicoQuant TCSPC module, and also with a Becker and Hickl TCSPC module). The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during the experiment constitutes a very powerful technique to quantitatively study protein interactions in live samples.
authors.affiliation: Institut de Génétique et Développement de Rennes, UMR 6290, CNRS, Université de Rennes 1, Rennes, France.
authors.forename: Sergi
authors.initials: S
authors.lastname: Padilla-Parra
authors.forename: Nicolas
authors.initials: N
authors.lastname: Audugé
authors.forename: Maïté
authors.initials: M
authors.lastname: Coppey-Moisan
authors.forename: Marc
authors.initials: M
authors.lastname: Tramier
mesh_terms: D004058:Diffusion; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D011485:Protein Binding; D060066:Protein Interaction Maps; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins